On the voltage‐dependent Ca2+block of serotonin 5‐HT3receptors: a critical role of intracellular phosphates

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Abstract
Natively expressed serotonin 5‐HT3receptors typically possess a negative‐slope conductance region in theirI–Vcurve, due to a voltage‐dependent block by external Ca2+ions. However, in almost all studies performed with heterologously expressed 5‐HT3receptors, this feature was not observed. Here we show that mere addition of ATP to the pipette solution is sufficient to reliably observe a voltage‐dependent block in homomeric (h5‐HT3A) and heteromeric (h5‐HT3AB) receptors expressed in HEK293 cells. A similar block was observed with a plethora of molecules containing a phosphate moiety, thus excluding a role of phosphorylation. A substitution of three arginines in the intracellular vestibule of 5‐HT3Awith their counterpart residues from the 5‐HT3Bsubunit (RRR‐QDA) was previously shown to dramatically increase single channel conductance. We find this mutant to have a linearI–Vcurve that is unaffected by the presence of ATP, with a fractional Ca2+current (Pf%) that is reduced (1.8 ± 0.2%) compared to that of the homomeric receptor (4.1 ± 0.2%), and similar to that of the heteromeric form (2.0 ± 0.3%). Moreover, whereas ATP decreased the Pf% of the homomeric receptor, this was not observed with the RRR‐QDA mutant. Finally, ATP was found to be critical for voltage‐dependent channel block also in hippocampal interneurons that natively express 5‐HT3receptors. Taken together, our results indicate a novel mechanism by which ATP, and similar molecules, modulate 5‐HT3receptors via interactions with the intracellular vestibule of the receptor.